DNA topoisomerases are enzymes that control and modify the topological states of DNA. This proposal aims at the elucidation of their biological roles and the molecular mechanisms by which they act. The methods of contemporary molecular biology will be applied. The possible involvement of the two known eukaryotic DNA topoisomerases in a number of processes, including gene expression, nucleosome assembly, chromosomal translocation, excision of integrated viral sequences, and replication, will be tested. Topoisomerase mutants will also be used to determine whether intracellular DNA in eukaryotic organisms is actively maintained in the underwound or negatively supercoiled state by the topoisomerases. Genetic and biochemical approaches will also be used to test whether the cellular target of a number of antitumor drugs is eukaryotic DNA topoisomerase II, and whether a protein kinase activity tightly associated with this enzyme is an intrinsic activity of the enzyme. Drug-resistant mutants of eukaryotic DNA topoisomerase II will be obtained to determine how these drugs enhance DNA cleavage by the topoisomerase. Studies of the roles of the two ATP-independent topoisomerases in bacterium, topoisomerase I and III, will be continued. Extensive mechanistic studies of both eukaryotic and prokaryotic DNA topoisomerases will be continued by the use of chemical, biochemical, and genetic analyses.